Identification of Dipeptidyl-Peptidase (DPP)5 and DPP7 in Porphyromonas endodontalis, Distinct from Those in Porphyromonas gingivalis

Abstract

Dipeptidyl peptidases (DPPs) that liberate dipeptides from the N-terminal end of oligopeptides are crucial for the growth of Porphyromonas species, anaerobic asaccharolytic gram negative rods that utilize amino acids as energy sources. Porphyromonas endodontalis is a causative agent of periapical lesions with acute symptoms and Asp/Glu-specific DPP11 has been solely characterized in this organism. In this study, we identified and characterized two P. endodontalis DPPs, DPP5 and DPP7. Cell-associated DPP activity toward Lys-Ala-4-methylcoumaryl-7-amide (MCA) was prominent in P. endodontalis ATCC 35406 as compared with the Porphyromonas gingivalis strains ATCC 33277, 16-1, HW24D1, ATCC 49417, W83, W50, and HNA99. The level of hydrolysis of Leu-Asp-MCA by DPP11, Gly-Pro-MCA by DPP4, and Met-Leu-MCA was also higher than in the P. gingivalis strains. MER236725 and MER278904 are P. endodontalis proteins belong to the S9- and S46-family peptidases, respectively. Recombinant MER236725 exhibited enzymatic properties including substrate specificity, and salt- and pH-dependence similar to P. gingivalis DPP5 belonging to the S9 family. However, the kcat/Km figure (194 µM-1·sec-1) for the most potent substrate (Lys-Ala-MCA) was 18.4-fold higher as compared to the P. gingivalis entity (10.5 µM-1·sec-1). In addition, P. endodontalis DPP5 mRNA and protein contents were increased several fold as compared with those in P. gingivalis. Recombinant MER278904 preferentially hydrolyzed Met-Leu-MCA and exhibited a substrate specificity similar to P. gingivalis DPP7 belonging to the S46 family. In accord with the deduced molecular mass of 818 amino acids, a 105-kDa band was immunologically detected, indicating that P. endodontalis DPP7 is an exceptionally large molecule in the DPP7/DPP11/S46 peptidase family. The enhancement of four DPP activities was conclusively demonstrated in P. endodontalis, and remarkable Lys-Ala-MCA-hydrolysis was achieved by qualitative and quantitative potentiation of the DPP5 molecule.

Figure 1. Cell-associated peptidase activity profiles of P. endodontalis and eight strains of P. gingivalis.

(A) Aliquots (5 µL) of bacterial cell suspensions (A₆₀₀ = 2.0) of P. gingivalis strains and P. endodontalis ATCC 35406 were incubated in a reaction mixture containing 20 µM Boc-Phe-Ser-Arg- or Z-His-Glu-Lys-MCA. (B) DPP activities toward Gly-Pro-, Lys-Ala-, Met-Leu-, and Leu-Asp-MCA were measured. Activity values are shown as the mean±S.D. (n = 3).

Figure 2. Alignment of amino acid sequences of P. endodontalis MER236725 and P. gingivalis DPP5.

The amino acid sequences of putative P. endodontalis DPP5 (PeDPP5, MER236725) and P. gingivalis DPP5 (PgDPP5, PGN_0756/MER034615) were aligned by Genetyx. Hyphens represent gaps introduced for maximal matching. Common amino acids are marked by asterisks. Three amino acids essential for serine proteases are written in red letters. Arrows indicate the N-terminal amino acid sequences determined with recombinant proteins.

Figure 3. Biochemical analysis of putative PeDPP5 (MER236725).

(A) Recombinant proteins (0.3 µg) of PgDPP5 (lane 1), putative PeDPP5 (lane 2), and aliquots (5 µL) of bacterial cell suspensions of P. gingivalis ATCC 33277 (lane 3), KDP136 (lane 4), and P. endodontalis (lanes 5 and 5C) were separated on SDS-PAGE gels, then stained with CB or subjected to immunoblotting (WB). The 100-kDa bands in lanes 5 and 5C were non-specifically observed with the alkaline phosphatase-conjugated second antibody. Lane M, molecular-weight markers. (B) NaCl-concentration dependence and (C) pH dependence of the hydrolyzing activity toward Lys-Ala-MCA of PgDPP5, putative PeDPP5 (MER236725), and P. endodontalis cells were determined. (D) The peptidase activities of recombinant PgDPP5 and PeDPP5 were determined using various dipeptidyl MCAs. Values are shown as the mean±S.D. (n = 3).

Figure 4. DPP5 mRNA and protein levels.

(A) Relative amounts of DPP5 mRNA in P. gingivalis ATCC 33277, KDP136, and P. endodontalis were measured by qPCR. Values are shown as the mean±S.D. (n = 3). *p<0.05; **p<0.01. (B) Inhibition ELISA was performed with recombinant PgDPP5 (white circles) and PeDPP5 (blue circles), as described in Materials and Methods. The amounts of DPP5 in P. gingivalis ATCC 33277, KDP136, and P. endodontalis whole cell lysates were determined to be 12.0±3.0, 20.9±3.5, and 51.8±12.8 ng/µg total proteins, respectively (mean±S.D., n = 3).

Figure 5. Alignment of amino acid sequences of P. endodontalis MER278904 and P. gingivalis DPP7.

The amino acid sequences of putative P. endodontalis DPP7 (PeDPP7, MER278904) and P. gingivalis DPP7 (PgDPP7, PGN_1479/MER014366) were aligned by Genetyx. Hyphens represent gaps introduced for maximal matching. Common amino acids are marked by asterisks. Three amino acids essential for serine proteases are written in red letters.

Figure 6. Biochemical analysis of putative PeDPP7 (MER278904).

(A) Recombinant PgDPP7 (lane 1), putative PeDPP7 (Leu³-Lys⁷¹⁰, lane 2), PgDPP11 (lane 3), and PeDPP11 (lane 4) (0.3 µg), and aliquots (5 µL) of cell suspensions of P. gingivalis ATCC 33277 (lane 5), KDP136 (lane 6), and P. endodontalis ATCC 35406 (lane 7) were separated on SDS-PAGE gels, then stained with CBB or subjected to immunoblotting (WB) against anti-PgDPP7 antiserum (300-fold dilution). Peroxidase conjugated anti-rabbit IgG (3,000-fold dilution) was used as the second antibody. Lane M, molecular-weight markers. (B) The hydrolyzing activities (mean±S.D., n = 3) of putative PeDPP7 (MER278904) and PgDPP7 were determined using dipeptidyl MCA.